Abstract

BackgroundBacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications.ResultsWe took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations.ConclusionThe Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

Highlights

  • Bacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited

  • Transposon-mediated BAC transgenesis is a new tool for precise delivery of BACs in both zebrafish and mice

  • SFtiagbulereTo2l2-BAC transgenesis in zebrafish and mice Stable Tol2-BAC transgenesis in zebrafish and mice. (a, b) Side views of double transgenic larvae (F1-18 and F1-43) carrying Tol2-Frevx1:Gal4-BAC (Tol2-BAC) and UAS:GFP that expressed GFP in spinal cord neurons at 2 dpf. (c) The insertion in F1-18 was located ~50 kb upstream of cng3. (d) The insertion in F1-43 was located in intron 7 of ntt4. 8 bp target site duplications are shown. (e) The structure of Tol2-BAC

Read more

Summary

Introduction

Bacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. BAC transgenesis has been carried out by microinjection of naked DNA into the cytoplasm of fertilized eggs in zebrafish [4,5] or oocyte pronucleus in mice [2,3] With these methods, integration of the BAC in the genome occurs at best in ~5-20% of the injected survivors in mice [3] and ~1-3% in zebrafish [5]. A recent extensive study showed that about half of BAC transgenic mice carried 1

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call